1. Field of the Invention
This invention relates generally to endocytosis of cell surface receptors and specifically to a novel protein which recognizes and binds to the endocytic code of the human insulin receptor and uses therefor.
2. Description of Related Art
Cell surface receptors have been broadly divided into class I receptors, which are constitutively located in clatharin coated pits under basal conditions, and class II receptors, which move from non-coated to coated regions of the plasma membrane when stimulated by ligand (Goldstein, et al., Ann. Rev. Cell Biol., 1:1-39, 1985). The preferential localization of class I receptor to coated pits is presumably dependent on the cytoplasmic domains of these receptors, as mutant receptors lacking this domain are randomly dispersed in the plasma membrane and poorly internalized. Several endocytic component receptors contain aromatic residues near the cytoplasmic face of the membrane, which are necessary for internalization. The NPVY.sup.807 sequence in the LDL receptor and the sequence Y.sup.20 TRF required for transferrin receptor internalization are predicted to form tight turns (Collawn, et al., Cell, 63:1061, 1990). Furthermore, the introduction of a tyrosine residue immediately distal to residues favoring turn formation in the cytoplasmic tail of glycophorin caused this molecule, normally excluded from coated pits, to be efficiently internalized (Ktistakas, et al., J. Cell Biol., 111:1393, 1990).
The internalization of class II receptors, which include the insulin and EGF receptors, is more complex. They require both intrinsic tyrosine kinase activity and specific amino acid sequences termed the endocytic codes (Russel, et al., J. Biol. Chem., 262:11833, 1987). The insulin receptor is a dimeric, transmembrane glycoprotein consisting of two extracellular .alpha. and two transmembrane .beta.-subunits.
The structure and function of the insulin receptor have been thoroughly analyzed after the cloning of the receptor cDNA (Ullrich, et al., Nature, 313:756, 1985). There are several structural domains within the receptor, a cysteine-rich domain in the .alpha.-subunit, a single transmembrane domain, and a region in the .beta.-subunit with homology to tyrosine-specific protein kinase (Kasuga, M., et al., Proc. Natl. Acad. Sci U.S.A., 80:2137-2141; Ullrich, A., et al., Nature, 313:756-761, 1985; Ebina, Y., et al., Cell, 40:747-758, 1985). The first step in insulin action involves high affinity binding of ligand to the receptor. This results in the activation of the tyrosine protein kinase located in the cytoplasmic domain of the .beta.-subunit of the receptor, autophosphorylation and phosphorylation on the tyrosine residues of certain cellular proteins, and internalization of the receptor (McClain, D., et al., J. Biol. Chem., 262:14663-14671, 1987; Hari, J., et al., J. Biol. Chem. 262:15431-15434, 1987). M-ophological studies have shown that the kinase deficient IRA.sup.1018 receptor does not enter coated pits in Rat-1 fibroblasts. A mutant receptor which has a deletion of an intact juxtamembrane region retains its insulin binding and insulin-stimulated tyrosine kinase activities but does not exhibit ligand-induced internalization. The ligand-stimulated internalization requires both receptor autophosphorylation and the intact juxtamembrane region which is encoded by exon 16 (Backer, J., et al., J. Cell Biol. 115:1535-1545, 1991; Thies, R., et al., J. Biol. Chem. 26,5:10132-10137, 1990). his region contains one copy of an NPXY sequence that is required for the endocytosis of class I receptors such as LDL-R and Tf-R.
Specific sequences involved in endocytosis of the human insulin receptor (hIR) have been analyzed by deletion and by point mutation. It was demonstrated that there were two independent sequences involved in endocytosis (Backer, J., et al., J. Cell Biol., 119:831-839, 1992; Rajagopalan, M., et al., J. Biol. Chem. 266:23068-23073, 1991). Cells containing a mutant hIR that have a deletion of the 16th exon which encodes 22 amino acids on the cytoplasmic side of the transmembrane region of the receptor .beta.-subunit, still bind insulin and activate as a tyrosine kinase. However, these cells are unable to internalize the hIR (Thies, et al., J. Biol. Chem., 265:10132, 1990). Therefore, the 16th exon encodes a domain necessary for ligand-dependent endocytosis.
Receptors mutated from GPLY to APLA internalized at only 32% of the rate of normal hIR. On the other hand, receptors mutated from NPEY to APEA internalized insulin at 87% of the normal rate. Similar to wild type receptors, both mutant receptors had the ability to bind insulin and undergo autophosphorylation. The information contained in the GPLY and, to a lesser extent, the NPEY sequences are necessary along with tyrosine kinase activity for signaling internalization of the insulin receptor.
Defects subsequent to ligand activation of insulin receptors have been identified as one cause of diabetes mellitus. Therefore, identification of cellular products that recognize and bind the endocytic code of the hIR and mediate endocytosis via that interaction may be essential. Such a cellular component would be a target for therapeutic compounds in order to alter the "trafficking" of the hIR and other cellular receptor molecules. The present invention provides a protein which binds to the functional endocytic code of hIR, thus fulfilling a longfelt need to identify cellular components which regulate hIR turnover.